Process for recovering vitamin b12



United States Patent PROCESS FOR RECOVERING VITAMIN B12 Henry M. Shafer, Westfield, N. 1., and Arnold J. Holland,

Penn Laird, Va., assignors to Merck & Co., Inc., Rah- Way, N. J., a corporation of New Jersey No Drawing. Application January 25, 1954, Serial-N0. 406,091

6 Claims. (Cl. 167-81) This invention is concerned generally with the recovery from solution of vitamin substances capable of promoting the growth of the microorganism Lactobacillus Iactis Dorner (LLD) and having animal protein factor (APP) activity. More particularly, it relates to a new and improved method for recovering vitamin B12 and/or vitamin Biz-like compounds (such as vitamin B128) from aqueous solutions, including fermentation broths containing the same, by adsorbing the vitamin B12 and/or vitamin Biz-like compounds from said solutions utilizing selected resinous adsorbents as the adsorbing agents.

This is a continuation-in-part of application Serial No. 186,115, filed September 21, 1950.

Vitamin B12 is a new chemical compound fully characterized in a co-pending application of applicants assignee, Serial No. 146,404, filed February 25, 1950, which is capable of promoting the growth of microorganism L. lactis Dorner, and which shows animal protein factor activity. It possesses marked and effective action in the therapeutic treatment of Addisonian pernicious anemia and other macrocytic anemias. Vitamin B12 is ordinarily prepared by fermenting an aqueous nutrient medium by means of a vitamin Biz-producing strain of Streptomyces and subjecting the fermentation broth thus obtained to purification operations.

By vitamin Biz-like compounds are meant the red crystalline compounds (not vitamin B12) which are obtained when a fermentation broth containing them is subjected to the purification treatment utilized in the purification of vitamin B12 (as described on pages 13 to 16 of said application, Serial No. 146,404) but omitting the counter-current distribution procedure employed as the final step in this purification operation. These vitamin Biz-like compounds can be characterized by the fact that they are readily convertible to pure vitamin B12 per se by treatment with cyanide ion, as described in a copending application of applicants assignee, Serial No. 120,009, filed October 6, 1949, now Patent No. 2,530,416.

The recoveryof vitamin B12 and/or vitamin Biz-like compounds from aqueous solutions, such as fermentation broths containing the same, has heretofore been accomplished by treating said broths with an adsorbent material such as activated charcoal or fullers earth which produces an adsorbate containing vitamin B12 and/or vitamin Biz-like compounds present in the broth together with various contaminants. The adsorbate is then eluted with a suitable solvent, such as an aqueous solution of pyridine or an alkyl substituted pyridine, and the eluate evaporated thereby producing an elaboration product which contains the vitamin B12 and/ or Biz-like compounds. The elaboration product thus obtained is extracted with an elaboration solvent, such as water or a lower aliphatic watermiscible alcohol, and the resulting extract is subjected to chromatographic fractionation by adding said extract to a column of adsorbent material and washing the column with solvent, fractionally to elute the vitamin B12 and/or vitamin Bm-like compounds from the adsorbent material.

The resulting eluate can then be further processed as set forth in the aforementioned application Serial No. 146,404 to produce pure vitamin B12, or to produce the vitamin Biz-like substances substantially free of contaminating materials.

Thus, the preparation of a vitamin-enriched extract suitable for chromatography, starting with a fermentation broth containing vitamin B12 and/ or vitamin Biz-lilte compounds, previously involved the following purification operations: 1) adsorption of the vitamin B12 and/or Biz-like compounds from fermentation broths utilizing activated carbon or fullers earth as the adsorbing agent; (2) elution of the adsorbate with pyridine; (3) evaporation of the pyridine .eluate; and (4) extraction of the elaboration product with another solvent.

While the foregoing procedure utilizing carbon as the adsorbing agent is successful in that it permits the recovery of vitamin B12 and/or vitamin Biz-like substances from fermentation broths containing the same, from the industrial point of view it is an expensive and objectionable method. Additionally, it has been found that unduly high losses of active materials occur in processing the eluates.

Attempts have been made, using batch operation, to utilize carbon as the adsorbent followed by elution of the resulting adsorbate with a two-phase water-butanol mixture. Fullers earth has also been used as an adsorbent for the LLB active components of fermentation broths but the use of fullers earth has necessitated the employment of batch procedures for both the adsorption and elution steps. Such batchwise procedures, although giving improved recoveries of vitamin B12 and like compounds, possess the objectionable features characteristic of batch operations and these procedures using carbon and fullers earth have not proved practicable for column operation.

Prior to the present invention, there was available no practical purification process employing a resinous adsorbent to remove vitamin B12 and/or vitamin Biz-like compounds. A number of resinous adsorbents were tried employing the usual adsorption conditions for each resinous adsorbent and all proved to be ineffective. For example, the followng representative adsorbents adsorbed little or none of the desired compounds: (a) a cation exchange resin deriving its exchange capacity from carboxylic groups; (b) a cation exchange resin deriving its exchange from sulphonic groups; (c) an anion exchange resin de riving its exchange capacity from secondary amine groups, and (d) an anion exchange resin deriving its exchange capacity from secondary and tertiary amine groups. In fact, resinous adsorbents of type (a) were found to selectively adsorb the anti-biotic streptomycin while rejecting substantially all of the vitamin B12 and/ or Biz-like compounds.

It is now discovered, however, that vitamin B12 and vitamin Biz-like compounds may be recovered from fermentation broths with surprising efficiency and facility utilizing as the adsorbent a substantially neutral porous decolorizing resin containing polar substituents in the form of hydroxyl or amino groupings, said resins being characterized as having little or no basic or acidic properties. These substantially neutral porous decolorizing resins (wherein the ion-exchange properties, if present, are decidedly in the background) constitute a wellrecognized class of resins which have been utilized for the adsorption of by-products in the purification of glucose as described in Dutch Patent No. 64,732, issued November 15, 1949. The properties and method of preparation of these substantially neutral porous decolorizing resins are more fully disclosed in Dutch Patent No. 59,449, issued June 16, 1947 and in U. S. Patent No. 2,389,865, issued November 27, 1945. We ordinarily prefer to utilize for this purpose an amine formaldehyde resin such as Permutit DR resin, manufactured by the Permutit Co., New York. This resin possesses a highly porous structure and contains polar substituents in the form of amino groupings. The resin has a low basic dissociation constant resulting from the amino groups and thus possesses only slightly basic properties. These substantially neutral porous decolorizing amine-formaldehyde resins can be prepared, for example, in accordance with the procedures set forth in illustrative Examples I and III of Dutch Patent No. 59,449, issued June 16, 1947. A1- ternatively, we prefer to employ a phenolic resinous adsorbent, such as Duolite 8-30 resin, manufactured by the Chemical Process Co., Redwood City, California. This resin is a porous decolorizing resin containing phenolic polar substituents. The resin has a very low acidic dissociation constant and thus shows only slightly acidic properties. These substantially neutral porous decolorizing phenolic resinous adsorbents can be prepared metal 1 carbonate and 0.1% alkali metal nitrite.

in accordance with procedures set forth in U. S. Patent As pointed out hereinabove, other resinous adsorbents (suchas the ionexchange resins) were found to beineifective for adsorbing vitamin B12 and/or vitamin Bizhlre compounds from aqueous solutions; Moreover, the herein-described neutral porous decolorizing resins containing polar substituents oifer substantial advantages from the standpoint of purity of product, recovery yields and economics of operation, over carbon and fullers earth, the only adsorbents previously utilized for adsorbi'ng vitamin B12 and'like compounds. Whereas, with the adsorbents, carbon or fullers earth, the adsorbing step serves merely as a concentration step after which nu sr u i c t Pur fi at o st p e qu r d, the-p uct obtained directly from fermentation broths utilizing a porous decolorizing' resinof the type described hereinabove as the adsorbent is relatively free fromundesirable materials present in the original broth. This is evidenced by thehigh ultimate recoveries of vitamin B12 which are obtained due to decreased losses in separating impurities in subsequent steps.

The vitaminBiz and .vitamin BlZ-llkC compounds are readily eluted in substantially quantitative yield from these novel resinous adsorbates utilizing ,a variety of aqueous organic solutions, particularly an aqueous acidic acetone solution, Not only does this elution procedure achieve substantially quantitative elution of the vitamin B12 and like compounds, but it accomplishes a still further. purification of the product due to the selective action of, the solvent which leaves adsorbed-impurities-in the resin. Moreover, the elutingsolvents which may be employedin the present procedure are readily recovered in contra'st to the exp'ensive and ,troublesomeurecovcry procedures, necessary. for recovering the pyridine previously utilized as the eluting solvent when carbon was employed. as the adsorbent material.

Ihe eluting solvents which we prefer toutilize in elutingthe vitamin B12 and/or vitamin Biz-like compoundsfrom the novel resin adsorbatesinclude a number ofaqueous organic solutions which maybe employed withyarying degrees-oflefliciency. Aspointed outhereinabove,ithe preferred .eluarits. are aqueous acetone solu- I tionsuacidified with a strong acid, that.is an acid having a dissociation constant for the first hydrogen of greater than, about, l We prefer to render the solution about 0.1 N with such an acid. vThe optimum acetone concentration lies between about 30 and 60%. Weak acids such as acetic acid may be employed but elution efficiency decreases. Instead of acetone wemay utilize a. number of organic solvents, such as other ketones, lower aliphatic alcohols esters, benzyl alcohol, and the .like,i1said organic solvents being at least partially miscible with; water. .Those solvents partially miscible with water are employed in amounts up to the limits of their solubilities inwa'ter. Solvents found to be of no value include toluene, benzene, chloroform, carbon tetrachloride and the like. Acid elution in the absence of a solvent is generally poor with low. removal, of active. substances. A number of. aqueous organic solvent solutions containing. no acid will elutemoderately well, including aqueous acetone, water-saturated with n-b-utanol as well as'aqueous ethylene glycol monoethyl ether. Some-elutionwill also take place withaqueous methanol, aqueous ethanol, aqueous ,ethyl acetate and with water saturated with benzyl alcohol,v although it is ordinarilyapreferred to employ. an acidic aqueous organic'solvent solution as the: eluant. M

Aqueous'solutions of strong baseswill also elute-the vitamin B12 and/or vitamin Biz-like compounds from the adsorbentbut such solutions also remove considerable amounts of the adsorbed impurities. It is therefore ordinarily preferred to elute the vitamin B12 component utilizing an aqueous organic acidic solvent solution. ..It has been found however, that aqueous solutions of weak bases'canbe employed to remove substantial amounts of adsorbedjmpurities while removing little or noneof the vitamin B12 and/or vitamin B12 like compounds. We

vhave found it useful to precede the elution of active substances by prewashing the adsorbate with a solution such as an aqueous solution containing about 1% alkali With this; added treatment, the eluates obtainedemployiug the preic r el ant ont in even. l s impu ies- The novel adsorption and elution procedure utilizing the herein disclosed, substantially neutral. porous. dfie. colorizing resins containingpolar substituents as the adsorbent can be employed generally for recovering LLD and APP-active substances from aqueous solutions. It is particularly useful for recovering vitamin B12 and vitamin Biz-like substances fromfermentittion broths produced by fermenting aqueous nutrient mediums'by means of selecteds'tr'a'ins of microorganisms of thesubphylum Fungi, whicharecapable of producing vitamin B 12 and/or vitamin Biz-like substances, and in particular strains: of Streptomyces, 'M ycobacterium, Pseudo'monas, Alternaria, and the like.

In carrying out the adsorption operation, where the resin adsorbate is utilized-for the first time, it has been found best to condition the resin prior to the adsorption operation. For example, when Du'olite' 8-30 is employed, the resin is placed in a column and washed with aqueous. alkaline solution. sodium hydroxide solution per .pound of resinis usually employed'and this-solution is-p'assed through the column at acontact time (volume of :resin in cc. divided by rate of flow in cc. per;minute) of about 25-30 minutes. Treatment of the Duoli'te 8-30 with: alkali causes the resin to changein'color from tan to purple. The alkaline wash is fellowed by .a water wash, about :two gallons per pound of resin at the same contact time. It has been foundadvantageousto follow the water wash with an acid wash, employing about 0.8 gallon of 2% aqueous hydrochloric acid solution per pound of-resin, and at a contact time of about fourteen minutes. Treatment of DuoliteS- 30 with acid causesthe resin to change incolor frompurple to fan. The acid wash ,is followed bya second water wash, about one gallon per pound of resin atacontact time of-fourteenminutes. Asecond alkaline wash, as described above, is given to the resin and this is followed by 1a water-wash at about 25-30 minutes contact time until the pH of the effluent is about 7-8. A's-noted hereinabove thisprelim inary treatment is referredtoas conditioning the resin. The resin .is then s'uitable for use in-adsorption. The resin pH is not important, since it adjusts quiekly' to that of :the solution being treated. g I

The-solution to be treated may be contacted .with the resinby batch miJring;,-butit is;muc h mo re advantageous to operate-by. passing the solutionthrou'gh acolumnof resin. 'The -.dire ction -,of flow through .the columnmay be either upward or downward. it appears to be preferable to' employ resin of particle size about 20-60 mesh for optimum hydraulic. efficiency. Fermentation-broth to'be treated is preferably at. a pH of about -58 :to avoid the-possibility of decomposition of active substances at higherand lower lpHs. The, pH isnot, however, critical as regardsthewlficiency .of adsorption.

As'is-usualin'most resin operations, considerable latitude is' allowable in the selection ofoperating conditions, such as quantities and flow rates. These conditions are both functions of the solutions treated and of economic considerations, muchas volumes to be processed laud equipment and space requirements. .Consequently at the-initiation ofoperations and/or when processingla new solution, a--certain amount of experimentation-is desirable forestablishing-optimal conditions. 'In general, the amount-ofsolution that can be processed with a given quantity of resin decreases as --;the-number of undesired substances present in the: solution increases. The types of undesired substances present is also a factor. {In adsorption,-i tmay besaid that the percentage of adsorption increases-with the contact time, up to a certain point,-an d 'resin require'mentsdeorease -withincreased contact :time. In. elution, eluantrequirements decrease-withincreased contact time, up to-a certain point, with corresponding increases in the concentration of activesubstancesin the eluates. r V

Inasmuch as the broths produced by=the fermentation of :Streptomycesgriseus are presentlyamong the..most.

important of ;the solutions containing vitamin-312 and/or vitamin. Biz-like compounds, the preferred conditions in the treatment of s'uch a broth with Duolite"S'30 are set forth as illustrative. "It will be understood thatlthe'se conditions can be varied considerably, and yet provide a practicable,process. S. griseizs: fermentation, broth as p'gesesn prepared ordinarily-exhibits on' the. order of 2,(l00':- 1 0,'0O0 uriits per, cc. oftLLD activity, assayed employingL: liz'c'tis'Dorner as a t'est'brga'n ism and vitamin About one gallon of 2% aqueous -In column operation',

was

B12 as a standard. For most eflicient operation, it is preferred to employ in column operation a feed volume of about 45-50 gallons of broth per pound of resin at a contact time of about ten minutes (a flow rate of about 0.026 gallon per minute per pound of resin; regenerated Duolite S-30 resin has a bulk density of about 3.86 pounds per gallon). Under these conditions and employing broth of about 6000 units per cc. of LLD activity, there results about 20-25% cumulative breakthrough (LLD activity remaining in the column efiluent). The active substances in the eflluent from the column are recovered by passing the effluent through a second resin column. Alternatively, two or more columns can be operated in series, alternately eluting and regenerating individual columns as they approach saturation. After adsorption, the column is washed with about 1.5 gallons of water per pound of resin at a contact time of about ten minutes. The wash displaces the last of the broth and removes highly pigmented impurities which may interfere in subsequent purification operations. Alternatively, adsorption and displacement of broth with water is followed 'by a column wash with about 0.5 gallon per pound of resin, of an aqueous solution containing 1% alkali metal carbonate and 0.1% alkali metal nitrite, at a contact time of about sixty minutes. This wash removes pigmented impurities and desorbs the majority of the remaining colored impurities, rendering them elutable with water. The column is then washed with about 2.5 gallons of water per pound of resin at a contact time of about ten minutes.

In eluting the foregoing adsorbate, we generally employ for elution about one gallon per pound of resin of the preferred eluant, acidified aqueous acetone, at a contact time of about 20-60 minutes. The quantity of eluant necessary is determined by observing the elution: after the water is displaced from the column, a light yellow or brown forerum is collected until the eluate becomes dark, red-black. This color signals the start of rich eluate which is collected separately until the color changes to light yellow or brown, when the tail cut starts and is collected separately. (A similar eluate color change occurs in the elution of Permutit DR from yellow, to brownish-red to yellow.) With the preferred eluants, it has been found that only negligible amounts of active substances are present in the forerun and tail cut, so that only the rich eluate need be utilized. The last of the eluant is displaced from the column with water.

The resin is then regenerated by washing with an aqueous alkaline solution. We prefer to wash the column with about one gallon per pound of resin of 2% sodium hydroxide solution, at a contact time of about 2530 minutes. The column is then washed with water until the pH of the effluent is about 7-8, at which point the column is ready for re-use in adsorption.

The following examples illustrate methods of carrying out the present invention, but it is to be understood that these examples are given by way of illustration and not of limitation.

' Example 1 In each of two 2" I. D. glass columns connected in series was placed 2500 .cc. of 20-60 mesh Duolite S-30 resin, a neutral porous decolorizing phenolic resinous adsorbent having practically no ion-exchange capacity and containing phenolic polar substituents, which can be prepared in accordance with the procedure set forth in U. S. Patent 2,389,865. The resin was being used for the first time for adsorption of LLD-active substances and was previously conditioned with alternate alkali and acid washes, finally being washed with water to pH 4-5, after an acid wash.

One hundred gallons of streptomycin fermentation broth at pH 6.2, previously treated to remove streptomycin, was assayed using L. lactis Dorner as the test organism and vitamin B12 as the standard and was found to have about 5800 units per cc. of activity (11,000 units-1 microgram of vitamin B12), or a total of about 21.9)( LLD units of activity. 'The broth was passed in upward fiow through the two columns in series, at an overall contact time of about twenty minutes (about 250 cc;/min.). The last of the broth was displaced from the column with water. The efliuent was assayed and found to contain a total of about 2.8 10 units of LLD activity. Thus, about 19.1)(10 units of activity (87.2%) were adsorbed on the columns. The columns were then washed in series with about fifteen liters of water at an overall contact time of about twenty minutes. The wash was assayed and found to show negligible LLD activity.

Each column was then eluted separately with 40% water-60% acetone (by volume made 1.0 normal with hydrochloric acid, at a contact time of about twenty minutes. About 6100 cc. of rich eluate was collected from the first column and about 6900 cc. from the second column. The total activity in the rich eluate cuts was about 18.7 10 units of LLD activity, about 98% of that adsorbed.

Example 2 In each of two 1%" I. D. glass columns connected in series was placed 100 cc. of 20-60 mesh Duolite S-30 resin, at neutral porous decolorizing phenolic resinous adsorbent having practically no ion-exchange capacity and containing phenolic polar substituents, which can be prepared in accordance with the procedure set forth in U. S. Patent 2,389,865. The resin had previously been used for adsorption of LLD-active substances and was afterwards regenerated by washing with alkali, acid and water to pH 45.

Six liters of streptomycin fermentation broth at pH 6.3, previously treated to remove streptomycin, was assayed using L. lactis Dorner and was found to have about 8200 units per cc. of activity, or a total of about 49.2 10 LLD units of activity. To the broth was added 0.75 mg. of radioactive vitamin B12a, giving 18,000 counts per minute, measured with a radiation counter. The total LLD activity after this addition was about 55.1)(10 units.

The broth was then passed in downward flow through the two columns in series, at an overall contact time of about 3540 minutes (about 5-6 cc./min.). The last of the broth was displaced from the column with water. The effluent was assayed for LLD activity, and its radioactivity Was measured. The results showed that 95% of the LLD-active substances and 91% of the radioactive material were adsorbed on the columns. The columns were then washed in series with about 2 /2 liters of water at an overall contact time of about twenty minutes.

Each column was then eluted separated with 40% water-60% acetone (by volume) made 0.1 normal with hydrochloric acid, at a contact time of about 60-75 minutes. The volume of eluant for the first column was about 600 ml., of which about 315 ml. was collected as rich cut. The volume of eluant for the second column was about 500 ml. of which about 200 ml. was collected as rich cut. The rich cuts were assayed for LLD activity, and their radioactivities were measured. The results showed that substantially all of the LLD-active and radioactive substances adsorbed were removed in the rich cuts. The ratio of activity found in the rich cut from the first column to activity found in the rich cut from the second column was about 10 tel for each type of activity.

When the above procedure was repeated employing streptomycin fermentation broth to which had been added radioactive vitamin B12, it was likewise found that the absorption and elution efficiencies were high, both when calculated on the basis of LLD activity measurements and on the basis of radioactivity measurements. Thus, it was found that this procedure is very effective for separating LLD-active substances, and in particular vitamin B12 and the vitamin Biz-like compound, vitamin Bus, from fermentation broths.

Example 3 Fifty gallons of streptomycin fermentation broth (pH 6.2), from which the streptomycin had been removed and having a total LLD activity of 5.5)(10 units, was passed through 3900 cc. of Duolite S-30 resin, a neutral porous decorolizing phenolic resinous adsorbent having, practically no ion-exchange capacity and containing phenolic polar substituents, which can be prepared in accordance with the procedure set forth in U. S. Patent 2,389,865 (pH 8), placed in one 1" column (1400 cc.) and one 2" column (2500 cc.), in series and in downward flow. The overall contact time was about 28 min utes. After water washing. about 75% of the activity was found to have been retained on the columns.

Each column was then eluted separately vwith 40% water60% acetone at a contact time of about twenty minutes. About 2150 cc. of rich eluate was collected mete-s 7 I from ithef.first eolumnarid about 1340 cc. from 1118;866- .ond,column. The total activity, in the richeluate cuts was about 9.5% :of that adsorbed.

v *Example 4 LEive gallons of -.s trepto rnycin -fer'rnentation broth (pH 7.4 ;from which :the streptomycin had been removed and/having ,a total .LLDactivity of T1.-1;. -1,0 ,u nits, was passed. through 154- cc; of Duol ite' S60; resin, a :neutral porous lde colorizing ,phenolic nesinons 1 adsorbent having practically no ion-exchange capacity and containing phenolic polar substituents which-can be prepared in accordance -with the procedure set forth in U. S. Patent 2,389,865 {(pH 8) placed-in a "1" ,icolumn, in downward flow. The-contact time was about5% minutes. After water washing, a about 73% of-the activity was found "to have b'een retained -on-the. column. 1

"The column was then eluted with water saturated :With N-butanol'zara contact timeof about .elevenminutes. About 950 cc. Of'richeIHate-WaS collected from the column. The: activity in therich eluate cut was about 55% of-that adsorbed.

Example; 5

' Threed" columnsjeach containing-154cc. .ofifDuolite S.'j3 0 ,resin, a neutralporous decolorizing phenolieresinous adsorbent'having practically no ion-exchange capacity and containing phenolic-polar substituents, which v'can' be preparedinjaccordance with the procedure set forth in ;U. .S. latent 2,389,865, (pH.8) lwere-arranged inqparallel. A separate five-gallon quantity 'of streptomycin fermentation broth, (pH 6-5 from which the streptomycin'fhad been removedv and having aitotalLLDactivity of 522)(10 units, waspassed through each column in downward'jfiow. The .c'ontact'timeineach case wasabout 5 minutes. ,Afterlwater washing, about 84%, 82% .and 82% of .the activity was found to;have been retained on ,columns'l, 2.and3, respectively.

jEachco'lumn was then eluated with a difierent solution, No. '1 withi40,% water-60% .acetone made 0.1 .normal withhydrochloric acid,.No. 2 with water saturatedwith n-butanol made 0.1 normal with ,hydrochloric .acid, and No. 3 with "40% ;water.,60% acetonemade 10.1 normal with phosphoric acid. Thecontact'timein each case was abouttenminutes. About 455.cc., 620 66921111 365 cc. of .rich 'eluate was collectedifrom' columns .1, 2 and' 3, respectively. The 'activity in the ,rich-eluate cut was about96'%,'78% and 9,8%.of.that adsorbed forQQlumns 1,2 and'3, respectively.

,An additional run ,showed that -,elution with 70% water-30% acetone made .0.1 normal with phosphoric acid was about as. effectiveasthei above 40% water- 60% acetone-phosphoric: acid .elution.

.Exampleo I1wo ;,1" columns each containing ;154,cc.. f;D uolite S3 0 resin, .arneutral porous decolorizing phenolic.resinous adsorbent Lhaving practically no ion-exchange 1; capacl ty .and contalning phenolic polar= substituents, which I can .be .prepared in accordance with ,the procedure =;set

forth.in.-U.LS. Patent 2,389,865, (pH'E8) were arrangedin parallel- .Aseparat fi egalloniq t y fts r p y broth (pH 6.2) ,.-frorn which :the streptomycin had .been removed tandihaving@atotalZLLD activity of 6.1)(10' 7 units, was passed through each-columnin downward flow.

The contact time in each case was about five minutes. After water washing, about 84% of the activity was foundzto. haveibeenretained on each of columns 1' and 2.

ecolurnn No. g1 wastheneluted with 40% water-60% acetonemade 0.1 normal with sulfuric acid, and. column Example 7 Fermentation broths "were ;prepared .*by :propagating three additional representative members of the j Fungi capable .of .producing LLD and APF- active substances: (1) Mycobacterium smegmatin (2.) fPseudomonas; turnichro'm'a (a new species of 'Pseudomonas capable of oxi- ,cies. EachIbrothataboutpH. ;7 was passed indownward flow-through a 1- /8" column. containing cc. of pno- 1ite S-30resin, ,a neutralporons decolorizingphenolic resinous adsorbent having practically .no ion exchange capacity andcontafiliflgphenolis polar substituents, which can be prepared in accordance with he. procedure set .forth in ULS. Patent 2,389,865,;atab0utpI-I 5. Theresin ,had previously been regenerated by washing, with alkali, .acid, and water. T he brothcontact time ineachcase-was about thirty-three minutes. The broth volumes' and tot al .LLD activitieswere: (1)3000 cc. and l0 .8 10 -unit s, .(2) 29.25 cc..-and :19.3 10 units,"and. (3-) 2625cc and 123x10 units. After water washing almost ;all of :the activity wasfound to have been retainedon the column ineach-case.

Each column was then eluted with 40% water-60% acetone made 0.1 normal with hydrochloricgacid ata contact time of about fifty minute's. Therich eluate volumes and approximatepercentagesof .adsorbed activity present in .the rieheluates were: (1) 250 cc. and'50-%, (2) 300cc. and 6 5%, and (3) 270 .cc. and 50%. (Optimum c n itions wer n stabl h r lf th s rothsi) Example 8 pared in accordance with the procedures set fOl'lhfiIl Dutch Patent 59.,449, at-pI-18. EFifteen ,litersof'streptomycin fermentation .brothat pH 7. 2, previously treated. to remove streptomycin, was assayedand was-found tohave about .2800-units per. cc. .ofLLLD :activity, or a,total of about.42 10 :units.of activity.; .The broth was passed in downward fiowthroughthe column'ofresim-at acontact time ofabout .tenminutes. *The broth was-followed bya water wash of 500 .cc. .flhe combinedbrotheflluent .and Wash contained a total-of about 5.2 10 units-Of activity. Thus-about. 36.8X10 'units of activity.(87 .5%) 'werea'dsorbed on thecolumn. i

T he. column was .then eluted with :40% water-1.60% acetone ,marle 0.1 normal with hydrochloric acid, atga contacttirne of about ;-twenty ;minutes. About .1080 cc. of rich eluate W-as collected and assayed. Thetotal activity in the rich. eluate was about 36.7)(10 {units of LLD activity, about 100% of that adsorbed.

Example 9 .Forty liters of streptomycin fermentation broth at pH 6.3, previously treated -.to remove streptomycin, was found on assay to have atotal of about 32 5)(10 LLD units-of activity, equivalent to, the activity of about*29.6 mg. of vitamin B12. To the brothwas added-4.34 mg. of. radioactivevitamin B12.

The broth was then adsorbed on and eluted: from 400 cc. of Duolite S-30 resin, aneutral porous decolorizing phenolic resinous adsorbent 'havingpractically no ionexchange capacity and containing phenolic polar substituents, which can beprepared in -accordancewithfth'e procedure set *forthiin' U. S1 Patent 2,3 89,865, in the manner described in Example 2. Rich eluate fractions were combined,adjuste'd to pH 7-8, concentrated and treated with potassium cyanide, as set forth in the aforementioned application, "Serial'No. 120,009, -to convert vitamin B12- Ex ple 1 0 :A -neutral porous -decolorizing phenolic resinous =a'd sorbent -wasprepared in-:accordance with a-procedure disclosed in- U. S.,Patent 2',389,86;5 "as follows: -A mixture of;-,-9 4 .g. ;of phenol, 1855 ml. of ;37% formaldehyde solution,'2.0 g. of sodium hydroxide and 68 g. of water was heated under reflux on the water bath for a period of about 100 minutes of which time a homogeneous gel had formed. The gel was maintained at a temperature of about 95 C. for a period of 2-3 hours and was then transferred to an autoclave fitted with a glass liner. The autoclave was flushed thoroughly with nitrogen, closed, and the autoclave and its contents were heated to a temperature of 118 C. over a 15 minute period, and maintained at a temperature of 118126 C. and equilibrium pressure for a period of about seventy minutes.

One-half gram of this cured resin was then mixed with a solution of 8.6 mg. of pure vitamin B12 in 10 n11. of water for a period of 30 minutes. The resin adsorbate was separated from the spent solution by centrifugation and washed with two 5 mL-portions of water. The water wash was added to the spent solution.

The resin adsorbate was eluted with one mL-portion and two 5 ml.-portions of an eluting solution consisting of 60% acetone and 40% of 0.1 N aqueous hydrochloric acid. The eluates were combined.

The vitamin B12 contents of the starting vitamin B12 solution, of the spent solution and washes, and of the combined eluates were determined by spectrophotometric measurement at 5500 A. The vitamin B12 adsorbed on the resin was calculated as the diflerence between that present in the starting B12 solution and that retained in the spent solution.

A comparison of these results with those obtained in a similar experiment using Duolite 8-30 resin is set forth in the following table:

Examination of the foregoing data shows that the vitamin B12 recovered by elution of the test resin adsorbate was about 58% of that originally present in the starting vitamin B12 solution as compared with a recovery yield of about 69% using the Duolite S-30 resin.

Example 11 A neutral porous decolorizing phenolic resinous adsorbent was prepared in accordance with a procedure disclosed in U. S. Patent 2,389,865 as follows: A mixture of 94 g. of phenol, 185 ml. of 37% formaldehyde solution, 2.65 g. of sodium hydroxide and 218 g. of water was heated under reflux on the water bath for a period of about 120 minutes at which time a homogeneous gel had formed. The gel was maintained at a temperature of about 95 C. for a period of 2-3 hours and was then transferred to an autoclave fitted with a glass liner. The autoclave was flushed thoroughly with nitrogen, closed, and the autoclave and its contents were heated to a temperature of 160" C. under a nitrogen pressure of 120-140 pounds per square inch, over a minute period. The contents of the autoclave were cooled to 125 C. in minutes and maintained at a temperature of 120-125 C and 120-140 pounds per square inch nitrogen pressure, for a period of about thirty-five minutes.

One-half gram of this cured resin was then mixed with a solution of 8.6 mg. of pure vitamin B12 in 10 ml. of water for a period of 30 minutes. The resin adsorbate was separated from the spent solution by centrifugation and washed with two 5 mL-portions of water. The water wash was added to the spent solution.

The resin adsorbate was eluted with one 10 mL-portion and two 5 mL-portions of an eluting solution consisting of 60% acetone and 40% of 0.1 N aqueous hyrochloric acid. The eluates were combined.

The vitamin B12 contents of the starting vitamin B12 solution, of the spent solution and washes, and of the combined eluates were determined by spectrophotometric measurement at 5500 A. The vitamin B12 adsorbed on the resin was calculated as the difference between that present in the original B12 solution and that retained in the spent solution.

A comparison of these results with those obtained in a similar experiment using Duolite S-30 resin is set forth in the following table:

Examination of the foregoing data shows that the vitamin B12 recovered by elution of the test resin adsorbate was about 85% of that originally present in the starting vitamin B12 solution as compared with a recovery yield of about 69% using the Duolite S-30 resin.

Various changes and modifications may be made in carrying out the present invention without departing from the spirit and scope thereof. Insofar as these changes and modifications are Within the purview of the annexed claims, they are to be considered as part of our invention.

We claim:

1. The process which comprises contacting an aqueous solution containing vitamin B12-active material with a substantially neutral porous decolorizing resin having substantially no ion-exchange capacity and containing polar substituents selected from the group consisting of hydroxyl and amino radicals thereby adsorbing said vitamin B12-active material on said resin, and contacting the adsorbate thus obtained with an aqueous solution containing a water-miscible organic solvent selected from the group consisting of ketones, alcohols and esters thereby eluting the vitamin B12-active material from said adsorbate.

2. The process which comprises contacting an aqueous solution containing vitamin B12-active material with a substantially neutral porous decolorizing phenolic resinous adsorbent having substantially no ion-exchange capacity and containing phenolic polar substituents thereby adsorbing the vitamin B12-active material on said adsorbent, and contacting the adsorbate thus obtained with an aqueous solution containing a water-miscible organic solvent selected from the group consisting of ketones, alcohols and esters thereby eluting the vitamin B12-active material from said adsorbate.

3. The process which comprises contacting an aqueous solution containing vitamin B12-active material with a substantially neutral porous decolorizing amine-formaldehyde resin having substantially no ion-exchange capacity and containing polar substituents in the form of amino groupings, thereby adsorbing the vitamin B12-active material on said resin, and contacting the adsorbate thus obtained with an aqueous solution containing a watermiscible organic solvent selected from the group consisting of ketones, alcohols and esters thereby eluting the vitamin B12-active material from said adsorbate.

4. The process which comprises contacting an aqueous solution containing vitamin B12 with a substantially neutral porous decolorizing phenolic resinous adsorbent having substantially no ion-exchange capacity and containing phenolic polar substituents thereby adsorbing the vitamin B12 on said adsorbent, and contacting the adsorbate thus obtained with an aqueous solution containing a watermiscible organic solvent selected from the group consisting of ketones, alcohols and esters thereby eluting the vitamin B12 from said adsorbate.

5. The process which comprises contacting an aqueous solution containing vitamin B12 with a substantially neutral porous decolorizing amine-formaldehyde resin having substantially no ion-exchange capacity and containing polar substituents in the form of amino groupings, thereby adsorbing the vitamin B12 on said resin, and contacting the adsorbate thus obtained with an aqueous solution containing a water-miscible organic solvent selected from the group consisting of ketones, alcohols and esters thereby eluting the vitamin B12 from said adsorbate.

6. The process which comprises contacting a substantially neutral porous decolorizing resin having substantially no ion-exchange capacity and containing polar substituents selected from the group consisting of hydroxyl and amino radicals with a fermentation broth containing vitamin B12-active material, thereby adsorbing said vitamin B12-active material on said resin, and contacting 

1. THE PROCESS WHICH COMPRISES CONTACTING AN AQUEOUS SOLUTION CONTAINING VITAMIN B12-ACTIVE MATERIAL WITH A SUBSTANTIALLY NEUTRAL POROUS DECOLORIZING RESIN HAVING SUBSTANTIALLY NO ION-EXCHANGE CAPACITY AND CONTAINING POLAR SUBSTITUENTS SELECTED FROM THE GROUP CONSISTING OF HYDROXYL AND AMINO RADICALS THEREBY ADSORBING SRAID VITAMIN B12-ACTIVE MATERIAL ON SAID RESIN, AND COPNTAVTING THE ADSORBATE THUS OBTAINED WITH AN AQUEOUS SOLUTION CONTAINING A WATER-MISCIBLE ORGANIC SOLVENT SELECTED FROM THE GROUP CONSISTING OF KETONES, ALCOHOLS AND ESTERS THEREBY ELUTING THE VITAMIN B12-ACTIVE MATERIAL FROM SAID ADSORBATE. 